Metabolic Effect of Short-Term Total Parenteral Nutrition Highly Enriched With Leucine or Valine in Rats Recovering From Severe Trauma EIGO MORI, PHD; MASAHARU HASEBE, MD; From The Trauma and Critical Care

of branched-chain amino acids as a supplement to total or enteral nutrition.1-3 BCAA-enriched TPN has been extensively utilized in liver diseases and severe trauma.4-6 However, there are also controversies about BCAA-TPN.7,8 An imbalance of amino acid composition in BCAA-enriched TPN may bring about some limitations to its longer-term utilization. The criteria for the optimal composition of BCAAs and their actual dosages are empirical and have been disputed.9-11 In addition, it has yet to be determined exactly what component of BCAA-TPN is responsible for its effectiveness. Similarly, the metabolic behavior of each BCAA in TPN remains obscure.9uo Hence, studies on metabolic behavior of individual BCAAs in TPN will offer much information for determining the most suitable quantity and composition of BCAAs in a TPN solution for a specific pathophysiologic situation. In the present study, we investigated the effect of alteration in the ratio of leucine (Leu) and valine (Val) in highly BCAA-enriched TPN (45% BCAA content) on the extent of their oxidation. Furthermore, the effect of BCAA-TPN having such an unbalanced composition for the recovery of metabolite levels of energy metabolism was examined after its short-term application in rats recovering from severe trauma.

compatible with the alterations of urinary nitrogen excretion, plasma Leu level, and metabolite contents of liver and was

muscle. The only difference in metabolite levels observed between the two TPN groups was in hepatic total adenine nucleotides. Plasma amino acid levels were largely unaffected by infusion of these TPN solutions highly enriched with branched-chain amino acids (45%), except for an approximately threefold elevation of the Val level in Val-TPN rats. Thus, when administered in a large quantity during such short-term TPN, Leu can exert its metabolic effect without causing an imbalance in plasma amino acids under severe catabolic conditions. ( Journal of Parenteral and Enteral Nutrition 16: 236-240, 1992)

MATERIALS AND METHODS

use

(BCAAs) has been practiced parenteral nutrition (TPN)

Reprint requests: Kunio Kobayashi, MD, The Trauma and Critical Care Center, Teikyo University School of Medicine, Itabashi, 173 Tokyo, Japan.

KUNIO KOBAYASHI, MD

Center, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

ABSTRACT. The metabolic impact of infusing a large amount of leucine (Leu) or valine (Val) was examined with regard to the corrective effect of total parenteral nutrition (TPN). Rats recovering from severe sepsis received either Leu- or Valenriched TPN solution for 30 hours. The in vivo behavior of the amino acids administered was explored by a pulse injection of 14 C-labeled Leu or Val. The recovery of 14 2 from Leu CO increased by 64% in the septic rats of Leu-TPN group (41% of p < .01), as compared with control rats receiving the same dose; TPN solution, whereas no significant rise in the 14 2 recovery CO from Val occurred in the septic rats given Val-TPN (45% of dose) in comparison with the corresponding controls. The enhancement of Leu catabolism to CO 2 in the Leu-TPN group

The clinical

AND

Materials

[14C]_L-Leucine and -valine (250 pci) were purchased from Du Pont-New England Nuclear Co (Boston). Enzyme preparations and coenzymes used in the metabolite assays were obtained from Boehringer-Mannheim GmbH (Mannheim,

Germany).

Induction of Sepsis and Treatment of Animals Male Sprague-Dawley rats weighing 190 to 220 g and obtained from Charles River Japan Inc (Atsugi, Japan) were fed and maintained as previously described.&dquo;

Throughout the study, we followed the guidelines for the care and use of laboratory animals set forth by the committee on animals of the Laboratory Animal Center of Teikyo University School of Medicine. Peritonitis was induced by cecal ligation according to the method of Ryan et al. 13 The details of the surgical procedures have been previously described.&dquo; The animals were returned to their cages and allowed water without nutrition sup-

port. About 16 hours later, the surviving animals (about 45% survival rate) were catheterized for TPN under anesthesia. The gangrenous cecum was dissected and removed at laparotomy. After closure of the abdomen, the rats were cannulated in the right external jugular vein with a Silastic catheter and placed in individual metabolic cages, as previously reported.12 The 20 rats were divided into two groups: Groups L and V received a Leu-enriched amino acid solution (Leu-TPN) and a Val-enriched solution (Val-TPN), respectively. Table I lists their compositions. The daily dosages were 1.3 g of 236

237

another 20 septic rats. After the removal of the cecum of the septic rats as described above, they were cannulated added at the usual dosages.12.14 The TPN solutions were and placed in individual closed-circuit metabolism chaminfused for 30 hours at a flow rate of 8.4 mL/h per bers for collection of the expired breath. These animals kilogram of body weight. Normal rats were cannulated received either of the two TPN solutions via the catheter and treated in similar manipulations including anes- for 30 hours (including the period of isotope experithesia, sham procedures for the operations that the septic ments). After 24-hour infusion of the TPN solution, a rats underwent, and laparotomy for sampling of liver pulse-dose of labeled BCAA was conducted by an intraand muscle tissues. They received either of the two venous injection of 5 ~Ci of L-14C-Leu (specific radioacBCAA-TPN solutions and served as the respective con- tivity, 348.0 mCi/mmol) or of L_14C- V al (specific radiotrols. They were designated as groups CL (n 6) and activity, 264.0 mCi/mmol) through the catheter. After CV (n one-shot administration, air was drawn through the 6). sealed glass unit using a pump, and the carbon dioxide in the expired breath was trapped in a mixture of monAnalytic Methods Arterial blood for amino acid analysis was drawn from oethanolamine-methylcellosolve. An aliquot (0.05 mL) the abdominal aorta. To measure the extent of altera- of the absorbent was taken at 30 and 45 minutes and tions in tissue metabolite levels in the operated septic then every hour for 6 hours. Infusion of the TPN solurats after TPN, the liver and the rectus abdominis muscle tions was continued via the catheter at an infusion rate of 2.0 mL/h until the time of death. The radioactivity were removed immediately after killing. The treatments of these samples for assays were carried out as described incorporated in protein was also measured after proteins, from the liver and the muscle by the procedure previously.&dquo; Standard assay methods using enzymatic extracted reactions were used to determine ATP,15 AD P , 16 AMP, 16 mentioned above, were dissolved in 0.1 N NaOH. Samanalyzed for 14C activity in 4.75 mL of the phosphocreatine,17 and glucose 6-phosphate.&dquo; The ples werescintillation fluid of Jeffay and Alvarez,23 using organic amounts of total protein and DNA were determined by a liquid scintillation spectrometer (Aloka LSC-703). the method of Lowry et all9 and of Ceriotti,2° respectively. Total RNA content was estimated from its absorbance Quenching was corrected for by the method of external standardization-channels ratio. The 12 sham-operated at 260 and 275 nm.21 The amount of hepatic glycogen rats, infused with either of the two BCAA-TPN solutions was determined colorimetrically by using anthrone reThe in the urine was deter- and treated in a similar manner, served as the respective samples agent.22 nitrogen mined by the micro-Kjeldahl technique. Amino acid anal- controls (groups CL and CV). yses were performed as previously reported.&dquo; The plasma amino acids of seven normal rats (group C), instead of Data Presentation those of the CL and CV groups, were used for compariAll values are expressed as means ± SD. Statistical son. comparison of groups CL, CV (C in the case of plasma amino acid contents), L, and V was based on the analysis Isotope Experiments of variance. With a significant F value, differences beThe efficiency of BCAA oxidation to C02 was esti- tween mean values were tested using a calculated wholly was considered signifimated in a separate series of experiments that used significant difference24; p < .01

nitrogen/kg of body weight and 200 kcal/kg of body weight. Electrolytes, trace elements, and vitamins were

=

=

cant.

Composition

TABLE I of branched-chain amino acid-total parenteral nutrition (BCAA-TPN) solutions (g/L)

RESULTS

Changes of Metabolite Levels

Distinct alterations in metabolite levels and in conprotein were seen in rats recovering from sepsis after administration of Leu-TPN or ValTPN (Table II). A rapid corrective effect of TPN was noted in hepatic energy metabolism because the values for these parameters were almost the same as those of the corresponding controls (CL and CV) and of normal rats.12,25 However, retarded recovery from the altered levels was still observed for hepatic energy charge and for muscle content of total adenine nucleotides, phosphocreatine, glucose 6-phosphate, RNA, and protein. The two types of BCAA-TPN applied exhibited similar corrective effects on the metabolic parameters during the recovery phase, except for the hepatic total content of adenine nucleotides. The rats given Val-TPN excreted more nitrogen into urine than did those give Leu-TPN; the excreted nitrogen (milligrams per 30 hours) during TPN was 380 ± 34

tents of RNA and

These two TPN solutions were isocaloric and isonitrogenous to each other. The proportion of BCAAs to the total amino acids was 45~ (w/ w) in both TPN solutions. The major nutritional difference between them was in the ratio of BCAAs.

in Liver and Muscle

238 TABLE II levels in liver and muscle

Changes of metabolite

~

~

~

~

~~

~~

~~

~

LGL, hepatic glycogen (mg/mg DNA); MPC, muscle phosphocreatine (~mol/mg DNA); G6P, glucose 6-phosphate. * CL, CV, L, and V denote control groups (CL, CV) and septic groups (L, V) given Leu-TPN or Val-TPN, respectively. t p < .01, significant difference us groups CL and CV. $ p ~ .01, significant difference us group L.

&dquo;

TABLE III

(10) for group L (p < .01 vs group CL), 521 ± 104 mg (10) for group V (p < .01 us group CV, p < .01 vs group L), 294 ± 30 mg (5) for group CL, and 284 ± 28 mg (6) mg

Uptake of ’4C radioactivity into protein fraction

for group CV.

Oxidation Studies The time courses of cumulative 14CO2 production in respiratory carbon dioxide were similar in septic rats and control rats (Fig. 1). Cumulative recovery (percent of dose) of 1¢C02 extrapolated to infinite time was as follows : group L, 41% (10), p < .01 vs group CL; group V, 45% (10); group CL, 25% (5); group CV, 42% (6). Because 1-14C-labeled BCAAs were not used in this study, any

attempt to quantify the oxidative rate of BCAA in vivo limited. The uptake of 14C into protein measured using the fractionated protein preparation from the liver was

and muscle is shown in Table III. Because the content

Values are means ± SD, shown as the percentage of of administered radioactivity per gram of protein. * p < .01, significant differences us the control group.

incorporated 1&dquo;C

of BCAAs differs from protein to protein and because not all the incorporated 14C species exist in the form of the injected BCAAs themselves, only the differences from the respective control values are meaningful. Amino Acid Concentrations That the plasma concentrations of many free amino acids decreased during the course of sepsis, showing

hypoaminoacidemia,

was

reported

in

our

previous

pa-

per.25 In addition, the

deviation of each amino acid from the normal level has become of interest in the use of these unbalanced TPN solutions. Figure 2 shows the improved profiles of plasma amino acid levels in septic rats given TPN. Most of the amino acid levels in plasma of the BCAA-TPN groups resembled those of the control rats. Although the amount of Val in the solution of ValTPN was almost twice that in the solution of Leu-TPN (Table I), the plasma Val level in Val-TPN rats increased threefold, as compared with that in Leu-TPN groups. Elevations of plasma glutamine and lysine levels and declines of arginine and proline levels were commonly observed in the septic rats given BCAA-TPN or conventional TPN during the recovery phase, 12 although a decrease

in threonine content

was

noted.

DISCUSSION

FiG. 1. Time course of 14C appearance in respiratory C02 in septic rats during administration of BCAA-TPN. L indicates the curve for septic rats receiving Leu-TPN (group L, n 10); V indicates the curve for those receiving Val-TPN (group V, n 10). CL and CV denote control rats receiving Leu-TPN (n 5) and Val-TPN (n 6), respectively. Each point represents the mean ± SD. =

=

=

=

The nutritional role and the actual catabolic process for each BCAA during administration of BCAA-TPN have been a matter of concern in regard to their clinical utilization. In our previous study comparing BCAA-TPN (BCAA content, 35.8% by weight) with conventional TPN (BCAA, 20.9%) in septic rats, the composition of

239

FIG. 2. Changes in plasma free amino acid levels in septic rats receiving BCAATPN. Columns C indicate the results for the normal rat group (n 7). This group did not receive TPN and differed from groups CL and CV. Columns L indicate the results for group L (n 9) and columns V for group V (n 9). *, p < .01, significant differences us normal group C; t, p < .01, significant differences us group L. =

=

=

BCAAs in BCAA-TPN was Leu:Val:Ile 2:1:1.12 In the plasma of the rats that received a large quantity of Leu the same traumatic evalua- during Leu-TPN (Fig. 2). The infused Leu appeared to model, study using present tion of the effects of modification in BCAA composition be actively degraded and effective in stimulation of muscle protein synthesis. This speculation was consistent on the initial corrective actions of BCAA-TPN was another aspect. The two TPN solutions with different with the observation that the increment of radioactivity proportions of Leu and Val used to this end had a 45% (0.05% of the dose) incorporated into 1 g of protein content of BCAAs. fractionated from the muscle tissues of the septic rats of BCAAs is more influenced receiving Leu-TPN, as compared with that of the group Whole-body degradation by glucose than by lipid,26 which suggests that the role CL rats, was much greater than the corresponding increof BCAAs as a fuel is important in some organs and that ment of radioactivity (0.01%) in Val-TPN rats (Table their catabolism is substantially linked with glucose and III). The effectiveness of BCAA-TPN for improvement energy metabolism. During BCAA-TPN, the presence of of muscle protein metabolism may be influenced by the BCAA composition as well as by their contents.’,10,&dquo; The an appropriate amount of Leu affects the degree of Val oxidation in vivo.27,28 The isotope experiment demon- metabolic alterations affected by Leu-TPN were also strated that Val was utilized more efficiently than Leu shown by significantly less urinary nitrogen excretion in the controls (groups CV and CL) as a substrate for and by the increase in total amount of adenine nucleoenergy production during simultaneous administration tides (Table II), as compared with that of the Val-TPN of glucose (200 g/L) (Fig. 1). Among BCAAs, only Val is group. The glucose 6-phosphate level appears to be perturbed solely glucogenic. Its overall catabolism proceeds without of the acid on undergoing stresses.33 No significant difintermediates of the tricarboxylic sensitively depletion cycle.29 Consequently, the maximal Val utilization al- ference between the two septic groups receiving BCAAready seen in the basal state was only slightly influenced TPN was observed in the glucose 6-phosphate level, the by the large amount of exogenous Val during Val-TPN. RNA/DNA ratio and the protein content of the liver and In contrast, septic stress increased Leu degradation by muscle tissues, or in the contents and composition of 64% above its basal oxidation level seen in the control muscle adenine nucleotides and phosphocreatine (Table rats (Fig. 1), similar to findings in starvation,30,31 diabeII). Nevertheless, the values of these metabolic parametes,31 and trauma.32 Leu or its keto-acid is not a signifi- ters were much better in both BCAA-TPN groups than cant fuel in resting skeletal muscle .30 However, the inthose obtained under conventional TPN in the previous crease of Leu oxidation to C02 observed in the septic study. 12 Restoration of most of the muscle parameters rats receiving Leu-TPN (Fig. 1) probably provided benmeasured to their respective control levels was retarded efits comparable with Val catabolism in the hepatic by 10% to 43% on the basis of mean values, regardless of the type of BCAA-TPN. The observation that plasma energy economy. The other results in the present study provided addi- glutamine still remained in a high level in the two TPN tional support for the finding on enhancement of Leu groups indicated that increased release of glutamine from catabolism under sepsis-induced catabolic conditions. In muscles was not effectively suppressed by application of contrast to the accumulation of plasma Val to approxithese TPN. The short term of TPN administration (30 mately twice the control level in the Val-TPN rats, the hours) was partly responsible for such delay in muscles. concentration of Leu was not appreciably increased in The initial and major site at which BCAA catabolism =

240 occurs

is skeletal muscle.29,31

Although no direct evidence

for BCAA effects on muscles was obtained in the present study, it seems reasonable to apply BCAA-TPN in enhanced catabolic situations where muscle protein is being wasted. The use of a highly BCAA-enriched solution during TPN inevitably involves a drawback of imbalance for amino acid supply. Our present results indicate that

such an adverse effect was absent after short-term administration of Leu-TPN. Therefore, if administered in early catabolic states, Leu-TPN can manifest an effective response to various physiologic demands by increasing the catabolic mode of Leu. ACKNOWLEDGMENTS

The authors express their appreciation to N. Iijima and H. Matsuda for technical assistance. This study was supported by a grant-in-aid for scientific research (60570602) from the Ministry of Education, Science, and

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